In-depth evaluation of state-of-the-art comprehensive extraction protocols for plant metabolomics using a classical validation approach

It is vital to pay much attention to the design of extraction methods for plant metabolomics, as any non-extracted or converted metabolites will greatly affect the overall quality of the study [1]. Method validation is however often omitted in plant metabolome studies, as the well-established methodologies for classical targeted analyses such as recovery optimisation cannot be strictly applied [2]. The aim of the present study is to thoroughly evaluate state-of-the-art comprehensive extraction protocols for plant metabolomics with liquid chromatography-photodiode array-accurate mass mass spectrometry (LC-PDA-amMS) by bridging the gap with classical method validation.

Validation of an extraction protocol in untargeted plant metabolomics should ideally be accomplished by validating the protocol for all possible outcomes, i.e. for all phytochemicals potentially present in the plant. In an effort to approach this ideal validation scenario, two plant matrices were selected based on their wide versatility of phytochemicals: meadow sweet (Filipendula ulmaria) for its polyphenols content, and spicy paprika powder (genus Capsicum) for its apolar phytochemicals (carotenoids, phytosterols, capsaicinoids) [3,4]. These matrices were extracted with comprehensive extraction protocols adapted from literature and analysed with a generic LC-PDA-amMS characterization platform that was previously validated for broad range phytochemicals analysis [3,5]. The performance of the extraction protocols was assessed based on extraction efficiency, repeatability and intermediate precision and on ionization suppression/enhancement evaluation.

The presentation will elaborate on the finding that none of the extraction methods allowed to exhaustively extract the metabolites. Furthermore, depending on the extraction conditions enzymatic degradation mechanisms can occur. Nevertheless, an overall good repeatability was observed for all extraction methods, which is essential for direct comparison between samples.

Acknowledgements: we thank the Flemish Fund for Scientific Research (FWO) to support this research (project 12M8315N).

Keywords: comprehensive phytochemicals extraction, plant metabolomics, generic LC-PDA-amMS platform, classical validation approach.

References:

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